Single-chain multivalent binding protein compositions and methods

Abstract

Provided are protein, nucleic acid, and cellular libraries of single chain multivalent binding proteins (e.g., scDVD and scDVDFab molecules) and methods of using these of these libraries for the screening of single chain multivalent binding proteins using cell surface display technology (e.g., yeast display).

We claim: 
     
         1 . A single chain multivalent binding protein having the general formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a first antibody heavy chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VH2 is a second antibody heavy chain variable domain, X2 is a linker, VL1 is a first antibody light chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VL2 is a second antibody light chain variable domain, and n is 0 or 1, and wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding sites. 
     
     
         2 . A single chain multivalent binding protein having the general formula (VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first antibody light chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VL2 is a second antibody light chain variable domain, X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VH2 is a second antibody heavy chain variable domain, and n is 0 or 1, and wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding site. 
     
     
         3 . The binding protein of  claim 1  or  2  which is a single-chain dual variable domain immunoglobulin molecules (scDVD). 
     
     
         4 . The binding protein of any one of the preceding claims, further comprising a cell surface anchoring moiety linked to the N and/or C terminus. 
     
     
         5 . The binding protein of  claim 4 , wherein the anchoring moiety comprises the Aga2p polypeptide. 
     
     
         6 . A polynucleotide encoding a binding protein of any one of the preceding claims. 
     
     
         7 . A host cell expressing a binding protein of any one of the preceding claims. 
     
     
         8 . A diverse library of binding proteins comprising a polypeptide chain having the general formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a first heavy chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VH2 is a second heavy chain variable domain, X2 is a linker, VL1 is a first light chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VL2 is a second light chain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding sites, and wherein the amino acid sequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently vary within the library. 
     
     
         9 . A diverse library of binding proteins comprising a polypeptide chain having the general formula (VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first antibody light chain variable domain, X1 is a linker with the proviso that it is not a constant domain, VL2 is a second antibody light chain variable domain, X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3 is a linker with the proviso that it is not a constant domain, VH2 is a second antibody heavy chain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and the VH2 and VL2 respectively combine to form two functional antigen binding sites, and wherein the amino acid sequences of VL1, X1, VL2, X2, VH1, X3, and/or VH2 independently vary within the library. 
     
     
         10 . The diverse library of  claim 8  or  9 , wherein each binding proteins further comprises a cell surface anchoring moiety linked to the N or C terminus. 
     
     
         11 . The diverse library of  claim 10 , wherein the anchoring moiety is a cell surface protein. 
     
     
         12 . The diverse library of  claim 10 , wherein the anchoring moiety is Aga2p. 
     
     
         13 . The diverse library of any one of the preceding claims, wherein the polypeptide chain is a scDVD. 
     
     
         14 . The library of any one of the preceding claims, wherein the amino acid sequence of at least one CDR of VH1, VH2, VL1 or VL2 independently varies within the library. 
     
     
         15 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR3 of VH1, VH2 independently vary within the library. 
     
     
         16 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR1 and HCDR2 of VH1 or VH2 independently vary within the library. 
     
     
         17 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH1 or VH2 independently vary within the library. 
     
     
         18 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR3 of VL1 or VL2 independently vary within the library. 
     
     
         19 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR1 and HCDR2 of VL1 or VL2 independently vary within the library. 
     
     
         20 . The library of any one of the preceding claims, wherein the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VL1 or VL2 independently vary within the library. 
     
     
         21 . The library of any one of the preceding claims, wherein X1 independently varies within the library and wherein X1 is selected from the amino acid sequences set forth in  FIG. 2 . 
     
     
         22 . The library of any one of the preceding claims, wherein X2 independently varies within the library and wherein X2 is (G 4 S)n, where n=1-10. 
     
     
         23 . The library of any one of the preceding claims, wherein X3 independently varies within the library and wherein X3 is selected from the amino acid sequences set forth in  FIG. 2 . 
     
     
         24 . The library of any one of the preceding claims, wherein the library of binding proteins share at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 amino acid sequence identity with a reference binding protein. 
     
     
         25 . The library of any one of the preceding claims, wherein VH1 and VH2 of the reference binding protein specifically bind to different antigens. 
     
     
         26 . A diverse library of polynucleotides encoding the diverse library of binding proteins of any one of the preceding claims. 
     
     
         27 . A diverse library of expression vectors comprising the diverse library of polynucleotides of  claim 26 . 
     
     
         28 . A library of transformed host cells, expressing the diverse library of binding proteins of any one of the preceding claims. 
     
     
         29 . The library of transformed host cells of  claim 28 , wherein the binding proteins are anchored on the cell surface. 
     
     
         30 . The library of transformed host cells of  claim 28 , wherein the binding proteins are anchored on the cell surface through Aga1p. 
     
     
         31 . The library of transformed host cells of  claim 28 , wherein the host cells are eukaryotic. 
     
     
         32 . The library of transformed host cells of  claim 31 , wherein the host cells are yeast. 
     
     
         33 . The library of transformed host cells of  claim 31 , wherein the yeast is selected from the group consisting of  Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe  and  Yarrowia lipolytica.    
     
     
         34 . The library of transformed host cells of  claim 31 , wherein the yeast is  Saccharomyces cerevisiae.    
     
     
         35 . A method of selecting a binding protein that specifically binds to a target antigen, the method comprising:
 a) providing a diverse library of transformed host cells expressing the diverse library of binding proteins of any one of  claims 8 - 25 ;   b) contacting the host cells with the target antigen; and   c) selecting a host cell that bind to the target antigen, thereby identifying a binding protein that specifically binds to a target antigen.   
     
     
         36 . A method of selecting a binding protein that specifically binds to a first and a second target antigen simultaneously, the method comprising:
 a) providing a diverse library of transformed host cells expressing the diverse library of binding proteins of any one of  claims 8 - 25 ;   b) contacting the host cells with the first and second target antigen; and   c) selecting a host cell that bind to the first and second target antigen, thereby identifying a binding protein that specifically binds to a first and a second target antigen simultaneously.   
     
     
         37 . The method of  claim 35  or  36 , wherein host cells that bind to the first and/or second antigen are selected by Magnetic Activated Cell Sorting using magnetically labeled antigen. 
     
     
         38 . The method of  claim 35 ,  36 , or  37 , wherein host cells that bind to the first and/or second antigen are selected by Fluorescence Activated Cell Sorting using fluorescently labeled antigen. 
     
     
         39 . The method of any one of  claims 35 - 38 , further comprising isolating the binding protein-encoding polynucleotide sequences from the host cells selected in step (c). 
     
     
         40 . A method of producing a binding protein, comprising expressing in a host cell a binding protein that was selected using the methods of any of  claims 8 - 25 . 
     
     
         41 . A method of producing a diverse library of binding proteins that specifically binds to a target antigen, the method comprising:
 a) providing a first diverse library of scDVD molecules, wherein the amino acid sequence of a first region of the scDVD molecules is varied in the library, and wherein each member of the library binds to the target antigen;   b) providing a second diverse library of scDVD molecules, wherein the amino acid sequence of a second region of the scDVD molecules is varied in the library, and wherein each member of the library binds to the target antigen;   c) recombining the first and second libraries to produce a third diverse library of scDVD molecules, wherein the third library comprises the first regions from the first library and the second region from the second library, thereby producing a diverse library of binding proteins that specifically binds to a target antigen.   
     
     
         42 . The method of  claim 41 , wherein the first and second libraries are recombined by yeast gap repair of polynucleotides encoding the libraries.